REGULATION OF TISSUE FACTOR PATHWAY INHIBITOR EXPRESSION

这些研究的主要目的是研究 诱导型组织因子途径抑制物（TFPI）基因表达 单核细胞和成纤维细胞。 采用北方印迹分析和TEPI 在这些试验中，申请人先前已经证明，内皮是 TFPI合成的主要生理位点，单核细胞和 成纤维细胞通常不表达TFPI或表达极少的TFPI。 但 申请人的初步数据表明，TFPI表达大大增加了 在粘附单核细胞和单核细胞U93细胞中上调。 在这 根据建议，申请人将在 TFPI基因在单核细胞和U937细胞中上调TFPI表达。 的 申请人还发现成纤维细胞显著上调TFPI 表达，并在此建议中，她计划定义 血清反应元件上调其在成纤维细胞中的表达。 初步数据还表明粘附单核细胞和U937细胞， 血清刺激的成纤维细胞和几种转化的细胞系， 表达TFPI同时表达加塔-2转录因子。 胶内迁移率 转移测定，来自HepG 2细胞的核提取物的因子（转化的 同时表达TFPI和加塔-2转录因子的细胞系）出现 以结合来自TFPI启动子区的DNA片段，所述DNA片段含有 推定的加塔基序。 另外，加塔-2转录因子反义 低聚物显著降低TFPI和加塔-2的表达 在贴壁UJ 937细胞中的转录因子mRNA。 因此加塔-2 TFPI基因表达可能需要转录因子。 这 可能性将在单核细胞和成纤维细胞中进一步测试， 有义和反义策略。 在申办者Paul的指导下生成初步数据 他是凝血和血栓形成领域的专家。 的 申请人将继续在Paul Bajaj的实验室工作， 建议的研究。 他也会不断地指导自己， 申办者Joel Bossenberg是一位经验丰富、公认的分子 生物学家 申请人将经常与咨询委员会会面 (Drs.”（《明史》卷100）“以其道，以其 中求进工作总 因此， 申请人进行拟议的研究。 预计该 研究将产生重要的新信息的调控诱导 TEPI基因在炎症反应中的表达

The primary goal of these studies is to investigate the regulation of inducible tissue factor pathway inhibitor (TFPI) gene expression in monocytes and fibroblasts. Using Northern blotting analysis and TEPI assays, the applicant had previously demonstrated that endothelium is the primary physiologic site of TFPI synthesis and that monocytes and fibroblasts normally express none to very little TFPI. However, the applicant's preliminary data indicate that TFPI expression is vastly upregulated in adherent monocytes and monocytic U93 cells. In this proposal, the applicant will define the adhesion response elements in the TFPI gene that upregulate TFPI expression in monocytes and U937 cells. The applicant has also found that fibroblasts significantly upregulate TFPI expression in response to serum and in this proposal she plans to define the serum response elements that upregulate its expression in fibroblasts. preliminary data also suggest that adherent monocytes and U937 cells, serum-stimulated fibroblasts and several transformed cell lines that express TFPI also express GATA-2 transcription factor. In gel mobility shift assays, factor(s) from nuclear extracts of HepG2 cells (a transformed cell line that expresses both TFPI and GATA-2 transcription factor) appear to bind to a DNA fragment from the TFPI promoter region containing a putative GATA motif. Additionally, GATA-2 transcription factor antisense oligomers significantly decreased the expression of TFPI and GATA-2 transcription factor mRNA in adherent UJ937 cells. Thus GATA-2 transcription factor may be required for TFPI gene expression. This possibility will be further tested in monocytes and fibroblasts by the use of sense and antisense strategies. Preliminary data was generated under the guidance of the sponsor, Paul Bajaj, who is an expert ina the field of coagulation and thrombosis. The applicant will continue to work in the laboratory of Paul Bajaj for the proposed studies. She will also receive constant guidance from the co- sponsor, Joel Eissenberg who is an experienced and recognized molecular biologist. The applicant will meet frequently with the advisory committee (Drs. Sly, Huang, Hyers, and Payvar) to seek guidance and discuss her progress. Thus ample resources and vast expertise is available to the applicant to perform the proposed studies. It is anticipated that the studies will yield important new information on the regulation of inducible TEPI gene expression during an inflammatory response.